J.L. Collier

The bovine mammary gland expresses multiple functional isoforms of serotonin receptors

Recent studies in dairy cows have demonstrated that serotonergic ligands affect milk yield and composition. Correspondingly, serotonin (5-HT) has been demonstrated to be an important local regulator of lactational homeostasis and involution in mouse and human mammary cells. We determined the mRNA expression of bovine 5-HT receptor (HTR) subtypes in bovine mammary tissue (BMT) and used pharmacological agents to evaluate functional activities of 5-HT receptors. The mRNAs for five receptor isoforms (HTR1B, 2A, 2B, 4, and 7) were identified by conventional real-time (RT)-PCR, RT quantitative PCR, and in situ hybridization in BMT. In addition to luminal mammary epithelial cell expression, HTR4 was expressed in myoepithelium, and HTR1B, 2A, and 2B were expressed in small mammary blood vessels. Serotonin suppressed milk protein mRNA expression (α-lactalbumin and β-casein mRNA) in lactogen-treated primary bovine mammary epithelial cell (BMEC) cultures. To probe the functional activities of individual receptors, caspase-3 activity and expression of α-lactalbumin and β-casein were measured. Both SB22489 (1B antagonist) and ritanserin (2A antagonist) increased caspase-3 activity. Expression of α-lactalbumin and β-casein mRNA levels in BMEC were stimulated by low concentrations of SB224289, ritanserin, or pimozide. These results demonstrate that there are multiple 5-HT receptor isoforms in the bovine mammary gland, and point to profound differences between serotonergic systems of the bovine mammary gland and the human and mouse mammary glands. Whereas human and mouse mammary epithelial cells express predominately the protein for the 5-HT7 receptor, cow mammary epithelium expresses multiple receptors that have overlapping, but not identical, functional activities.

Environmental physiology of livestock

Contributors ix Foreword xi 1 From Heat Tolerance to Heat Stress Relief: An Evolution of Notions in Animal Farming 1 Amiel Berman 2 Physiological Basics of Temperature Regulation in Domestic Animals 17 Donald E. Spiers 3 Heat Stress and Evaporative Cooling 35 Kifle G. Gebremedhin 4 Regulation of Acclimation to Environmental Stress 49 Kajal Sankar Roy and Robert J. Collier 5 Environment and Animal Well-Being 65 S. D. Eicher 6 Effects of Environment on Metabolism 81 Lance Baumgard and Robert P. Rhoads 7 Impact of Hot Environment on Nutrient Requirements 101 Umberto Bernabucci 8 Effects of Environment on Animal Health: Mechanisms and Regulatory Inputs 129 Ted H. Elsasser, Cong-Jun Li, Jessica Shaffer, and Robert J. Collier 9 Effect of Environment on Immune Functions 165 Nicola Lacetera 10 Strategies for Improvement of Thermal and Reproductive Responses under Heat Stress 181 David Wolfenson and William W. Thatcher 11 Prospects for Improving Fertility during Heat Stress by Increasing Embryonic Resistance to Elevated Temperature 199 Peter J. Hansen 12 Environmental Heat Stress Impairs Placental Function, Fetal Growth and Development, and Postnatal Performance in Livestock 209 Dustin T. Yates, Xiaochuan Chen, and Sean W. Limesand 13 Effects of Photoperiod on Domestic Animals 229 Geoffrey E. Dahl and Izabella M. Thompson 14 Rethinking Heat Index Tools for Livestock 243 J. B. Gaughan, T. L. Mader, and K. G. Gebremedhin 15 Strategies to Reduce the Impact of Heat and Cold Stress in Dairy Cattle Facilities 267 John R. Smith and J. P. Harner, III 16 Genotype by Environment Interactions in Commercial Populations 289 Ignacy Misztal and Peter Lovendahl 17 Responses of Poultry to Environmental Challenges 309 J. Brake and S. Yahav Index 337

A dose-response evaluation of rumen-protected niacin in thermoneutral or heat-stressed lactating Holstein cows

Twenty-four multiparous high-producing dairy cows (40.0±1.4kg/d) were used in a factorial design to evaluate effects of 2 environments [thermoneutral (TN) and heat stress (HS)] and a dose range of dietary rumen-protected niacin (RPN; 0, 4, 8, or 12g/d) on body temperature, sweating rate, feed intake, water intake, production parameters, and blood niacin concentrations. Temperature-humidity index values during TN never exceeded 68 (stress threshold), whereas temperature-humidity index values during HS were above 68 for 24h/d. The HS environment increased hair coat and skin, rectal, and vaginal temperatures; respiration rate; skin and hair coat evaporative heat loss; and water intake and decreased DMI (3.5kg/d), milk yield (4.1kg/d), 4% fat-corrected milk (2.7kg/d), and milk protein yield (181.7g/d). Sweating rate increased during HS (12.7g/m(2) per h) compared with TN, but this increase was only 10% of that reported in summer-acclimated cattle. Niacin supplementation did not affect sweating rate, dry-matter intake, or milk yield in either environment. Rumen-protected niacin increased plasma and milk niacin concentrations in a linear manner. Heat stress reduced niacin concentration in whole blood (7.86 vs. 6.89μg/mL) but not in milk. Reduced blood niacin concentration was partially corrected by dietary RPN. An interaction existed between dietary RPN and environment; dietary RPN linearly increased water intake in both environments, but the increase was greater during HS conditions. Increasing dietary RPN did not influence skin temperatures. During TN, supplementing 12g/d of RPN increased hair coat (unshaved skin; 30.3 vs. 31.3°C at 1600h) but not shaved skin (32.8 vs. 32.9°C at 1600h) temperature when compared with 0g/d at all time points, whereas the maximum temperature (18°C) of the room was lower than skin temperature. These data suggest that dietary RPN increased water intake during both TN and HS and hair coat temperature during TN; however, core body temperature was unaffected. Thus, encapsulated niacin did not improve thermotolerance of winter-acclimated lactating dairy cows exposed to moderate thermal stress in Arizona.

Alterations of A549 lung cell gene expression in response to biochemical toxins

Health risks associated with the inhalation of potentially toxic materials have been a topic of great public concern. In vitro cellular analyses can provide mechanistic information on the molecular-level responses of lung-derived cell lines to a variety of these hazards. This understanding may be used to develop methods to reduce the damage from such toxins or to detect early stages of their effects. Here we describe an evaluation of the alterations in gene expression of an immortalized lung cell line (A549, human type II epithelia) to a variety of inhalation health hazards including etoposide, gliotoxin, streptolysin O, methyl methansesulfonate (MMS), and Triton X-100. The A549 cells display a dose–response relationship to each toxin with initial responses including alterations in metabolic activity, increases in membrane permeability, and initiation of response genes. In general, membrane-damaging agents (streptolysin O and Triton X-100) induce production of new ion channel proteins, structural proteins, and metabolic enzymes. Gliotoxin impacted the metabolic machinery, but also altered ion channels. Etoposide and MMS caused alterations in the cell cycle, induced DNA repair enzymes, and initiated apoptotic pathways, but MMS also induced immune response cascades. The mechanism of cell response to each toxin is supported by physiological analyses that indicated a fairly slow initiation of cell response to all compounds tested, except for Triton, which caused rapid decline in cell function due to solubilization of the cell membrane. However, Triton does induce production of a number of cell membrane-associated proteins and so its effects at low concentrations are likely translated throughout the cell. Together these results indicate a broader array of cellular responses to each of the test toxins than have previously been reported.

Milk yield differences between 1× and 4× milking are associated with changes in mammary mitochondrial number and milk protein gene expression, but not mammary cell apoptosis or SOCS gene expression

Milking frequency is known to affect milk production and lactation persistence in dairy cows. Despite this, the mechanisms underlying this effect are only partially understood. Previous work in dairy cows examining increases in milk yield due to increased milking frequency have identified changes in apoptosis and expression of genes regulating cytokine signaling. In addition, changes in mitochondrial biogenesis and function have been suggested to play a role during the lactation cycle in regulating milk production. The goal of this study was to test the hypothesis that, when maintained over an entire lactation, extreme differences in milking frequency would be reflected in differences in apoptosis, mammary mitochondrial number, and the mammary expression of genes known to inhibit cytokine signaling. Primiparous Holstein cows (n=6) were assigned to the study 40d before parturition after which 1 udder half was milked once daily (1×) and the other 4 times daily (4×) Mammary biopsies were collected at 15, 60, 120, and 230d of lactation. Average milk yield from the 4× side was 3 times higher than from the 1× side. Analysis of milk composition revealed that protein, lactose, and solids-not-fat percentages were lower in 1× than 4× udder halves. Mammary cell apoptosis was not affected by milking frequency. Mammary cell mitochondrial number, as estimated by succinate dehydrogenase staining, was higher in early lactation, decreasing as days in milk increased, and with increased milking frequency. Although mammary expression of α-lactalbumin (LALBA) and β-casein (CSN2) was significantly increased in 4× glands, the expression of suppressors of cytokine signaling were similar between 1×- and 4×-milked halves. These results support the conclusion that changes in milk production in response to extreme differences in milking frequency may be related to alterations in mitochondrial number and lactose synthesis, but not apoptosis.

Evaluation of dietary betaine in lactating Holstein cows subjected to heat stress

Betaine (BET), a natural, organic osmolyte, improves cellular efficiency by acting as a chaperone, refolding denatured proteins. To test if dietary BET reduced the effect of heat stress (HS) in lactating dairy cows, multiparous, lactating Holstein cows (n=24) were blocked by days in milk (101.4±8.6 d) and randomly assigned to 1 of 3 daily intakes of dietary BET: the control (CON) group received no BET, mid intake (MID) received 57mg of BET/kg of body weight, and high dose (HI) received 114mg of BET/kg of body weight. Cows were fed twice daily and BET was top-dressed at each feeding. Cows were milked 2 times/d and milk samples were taken daily for analysis. Milk components, yield, feed intake, and water intake records were taken daily. Rectal temperature and respiration rate were taken 3 times/d at 0600, 1400, and 1800h. Cows were housed in environmentally controlled rooms and were allowed acclimation for 7d at thermoneutral (TN) conditions with a mean temperature-humidity index of 56.6. Cows were then exposed to 7d of TN followed by 7d of HS represented by a temperature-humidity index of 71.5 for 14d. This was followed by a recovery period of 3d at TN. Dietary BET increased milk yield during the TN period. No differences were found between BET and CON in total milk production or milk composition during HS. The increase in water intake during HS was not as great for cows fed BET compared with controls. The cows on CON diets had higher p.m. respiration rate than both MID and HI BET during HS, but lower rectal temperature compared with BET. No difference was found in serum glucose during TN, but cows given HI had elevated glucose levels during HS compared with CON. No differences were found in serum insulin levels between CON and BET but an intake by environment interaction was present with insulin increasing in HI-treated lactating dairy cows during HS. The heat shock response [heat shock protein (HSP) 27 and HSP70] was upregulated in bovine mammary epithelial cells in vitro. Blood leukocyte HSP27 was downregulated at the HI dose under TN conditions and HSP70 was upregulated at the HI dose and this effect was increased by HS. No effect was seen with the MID dose with HSP27 or HSP70. The lack of effect of BET at MID may be associated with uptake across the gut. We conclude that BET increased milk production under TN conditions and was associated with reduced feed and water intake and slightly increased body temperatures during HS of cows fed BET. The effect of BET on milk production was lost during HS with HI BET, whereas serum glucose levels increased during HS.

Effects of niacin and betaine on bovine mammary and uterine cells exposed to thermal shock in vitro

The objective of this study was to investigate the direct effects of feed supplements niacin and betaine on the heat shock responses of in vitro cultured cells derived from bovine mammary and uterine tissues. First, we determined the mRNA expression profiles of the niacin receptor (GPR109A) in bovine tissues (liver, skin, uterus, udder, and ovary) and in cells derived from bovine mammary epithelium (mammary alveolar cells, MAC-T; bovine mammary epithelial cells, BMEC) and endometrium (bovine endometrial cells, BEND). We found that GPR109A was distributed in all examined tissues and cells, and the highest expression was in cells from skin and udder. Second, we evaluated the effects of niacin treatment on the mRNA abundance of heat shock proteins 70 and 27 (HSP70 and HSP27) in MAC-T, BMEC, and BEND under thermoneutral conditions and heat stress, and whether these effects were associated with alterations in the mRNA expression of prostaglandin E2 synthesis-related genes, including cyclooxygenase 1 and 2 (COX-1 and COX-2) and microsomal prostaglandin E synthase 1 and 2 (mPGES-1 and mPGES-2). Quantitative PCR data indicated that niacin suppressed HSP70 mRNA expression in BMEC and both HSP70 and HSP27 in BEND under thermoneutral conditions. Only COX-2 expression was downregulated by niacin in BMEC; other prostaglandin E2 synthesis-related genes stayed unaltered in BMEC and BEND. The mRNA abundance of HSP70, COX-1, COX-2, and mPGES-1 were elevated in niacin-treated MAC-T. During heat stress, niacin increased mRNA levels of HSP70 and HSP27 in MAC-T and HSP27 in BEND, but decreased HSP70 in BMEC. Although mPGES-2 was stimulated by niacin in BEND, the mRNA expression of prostaglandin E2 synthesis-related genes were consistent with neither HSP70 nor HSP27 expression patterns in niacin-treated BMEC and MAC-T. These data suggest that the effects of niacin on heat shock protein expression and prostaglandin E2 synthesis were not well coupled in these cells. Finally, we tested the effects of betaine treatment on viability and apoptosis in BMEC. Compared with control cultures, viability was higher in betaine-treated cells at 8 h under thermoneutral conditions and at 16 h in heat stress, and apoptotic rates were lower at 8 h. Our data support a dual role for niacin in regulating heat shock protein expression in normal and heat-shocked cells derived from mammary and uterine tissues, and positive effects of betaine in regulating mammary cell viability during heat stress.

An evaluation of an immunomodulatory feed ingredient in heat-stressed lactating Holstein cows: Effects on hormonal, physiological, and production responses

Holstein cows (n = 30) were balanced by days in milk, milk production, and parity (91 ± 5.9 d in milk, 36.2 ± 2.5 kg/d, and 3.1 ± 1.4, respectively) and fed OmniGen-AF (OG; Phibro Animal Health, Teaneck, NJ), an immune stimulant, at 0 g/cow per d for control (CON) or 56 g/cow per d for OG for 52 d on a commercial dairy. At 52 d of the study cows were randomly selected (n = 12) from both groups (6 OG and 6 CON) and housed in environmentally controlled rooms at the Agricultural Research Complex for 21 d at the University of Arizona. Cows were subjected to 7 d of thermoneutral (TN) conditions, 10 d of heat stress (HS), and 4 d of recovery (REC) under TN conditions. Feed intake, milk production, and milk composition were measured daily. Rectal temperatures (RT) and respiration rates (RR) were recorded 3 times per day (600, 1400, and 1800 h). Blood samples were taken on d 7 (TN), 8 (HS), 10 (HS), 17 (HS), and 18 (TN) during the Agricultural Research Complex segment. Cows in HS had higher RR and RT and water intake and lower dry matter intake and milk yield than these measures in TN. There was a treatment × environment interaction with cows fed OG having lower RR and RT and higher dry matter intake during peak thermal loads than CON. However, milk yield did not differ between groups. Cows fed OG had lower milk fat percent than CON (3.7 vs 4.3%) during HS. The SCC content of milk did not differ between treatment groups but rose in both groups during the REC phase following HS. Plasma insulin and plasma glucose levels were not different between groups. However, plasma insulin in both groups was lower during acute HS, then rose across the HS period, and was highest during the REC phase. Plasma cortisol levels were highest in all cows on the first day of HS (d 8) but were lower in cows fed OG compared with CON. However, plasma ACTH concentrations were elevated in OG-fed animals at all times samples were collected. Plasma ACTH was also elevated in cows fed both OG and CON during HS. Feeding OG reduced plasma cortisol during acute but not chronic HS and increased basal plasma ACTH, suggesting that OG treatment may alter the hypothalamic pituitary adrenal axis.

Technical note: Method for isolation of the bovine sweat gland and conditions for in vitro culture

Apocrine sweat glands in bovine skin are involved in thermoregulation. Human, horse, and sheep sweat gland epithelial cells have been isolated and grown in vitro. The present study was conducted to identify a method to isolate bovine sweat glands and culture apocrine bovine sweat gland epithelial cells in vitro. Mechanical shearing, collagenase digestion, centrifugation, and neutral red staining were used to identify and isolate the apocrine glands from skin. Bovine sweat glands in situ and after isolation comprised 2 major cell types consisting of a single layer of cuboidal epithelial cells resting on a layer of myoepithelial cells. In situ, the glands were embedded in a collagen matrix primarily comprising fibroblasts, and some of these cells were also present in the isolated material. The isolated material was transferred to complete medium (keratinocyte serum-free medium, bovine pituitary extract, and human recombinant epidermal growth factor + 2.5% fetal bovine serum) in a T 25 flask (Falcon, Franklin Lakes, NJ) with media film and then incubated at 37°C for 24 h. After sweat glands adhered to the bottom of the flask, an additional 2 mL of complete medium was added and the medium was changed every 3 d. Isolated apocrine sweat glands and bovine sweat gland epithelial cells were immunostained for cytokeratin and fibroblast specific protein, indicating fibroblast-free cultures.